Eukaryotic mRNA with its 5-cap is usually of central importance for the cell. RNA (Fig. 2). Moreover, RNAs capped with those nucleotide analogues were translated even in the absence pHZ-1 of the capping enzyme Pifithrin-alpha supplier instead of GTP to generate RNAs with the respective caps . Co-transcriptional cappingIn co-transcriptional capping, cap analogues are added directly to the IVT. Their incorporation at the 5-end by RNA polymerases with relaxed substrate specificity (e.g., T3, T7 or SP6 RNA polymerases) directly yields the respective 5-capped mRNA (Fig. 3). Internal incorporation of cap analogues during IVT does not occur, because cap analogues lack a free 5-triphosphate. Open in a separate window Physique 3 Schematic representation of co-transcriptional capping with different cap analogues. A DNA-dependent RNA polymerase initiates transcription from a DNA template by incorporation of a cap analogue, thereby producing capped RNA. When m7GpppG is used as a cap analogue, miscapped RNA with the cap analogue incorporated in its reverse orientation is usually produced in addition to the correctly capped RNA. With the ARCA cap, reverse incorporation is usually excluded . The most commonly used cap analogue is usually m7GpppG but several modified or alternate cap analogues are also accepted by RNA polymerases. Therefore, this route can be used to install non-natural dinucleotides at the 5-end that are accessible for a further chemical reaction . One often Pifithrin-alpha supplier overlooked limitation of co-transcriptional capping is usually that not all mRNA obtained from IVT is usually capped, simply because the cap analogue competes with GTP as initiator nucleotide. Importantly, the ratio of capped/uncapped mRNA is usually not visible on a gel. This issue can be mitigated by lowering the GTP concentration or by digesting uncapped (i.e., triphosphorylated) RNA with a 5-polyphosphatase which produces monophosphorylated RNA followed by 5-phosphate-dependent exonuclease digestion. Another problem encountered with m7GpppG as initiator is usually elongation into the wrong direction, namely at the 3-OH of m7G, yielding mRNA with Pifithrin-alpha supplier the cap in reverse orientation (Fig. 3). Up to one half of the mRNA can contain the cap in its reverse orientation and will not be translated . This problem was solved by developing anti-reverse cap analogues (ARCA) that are methylated or deoxygenated at the 3-OH of the or m7GpppAN(1 9 nt), bacteriophage T7 gene 4 primase  or its active domain  can be used. Primase incorporates cap analogues exclusively in their correct orientation. Normally, gene 4 primase from the T7 phage produces short RNAs with the sequence pppAC from a DNA template. Matsuo et al. observed that GpppA or m7GpppA can be incorporated Pifithrin-alpha supplier as efficiently as ATP as the first nucleotide . The substrate specificity of gene 4 primase for adenosine as the first nucleotide prevents incorporation of GpppA in its reverse orientation and incorporation of GpppG altogether. This method was used for the production of isotope-labeled capped RNA for cap-eIF4E NOESY-NMR studies . Peyrane et al. demonstrated that using the N-terminal fragment bearing the primase activity resulted in comparable preparation yield for the RNA while expression and solubility of the fragment were improved . mRNA cap analogues Preparation of cap analoguesThe co-transcriptional capping described above requires the preparation of cap analogues which are added to the transcription reaction. Ideally, these cap analogues should meet the following criteria: (i) high incorporation efficiencies when added to IVT, (ii) correct orientation when incorporated into RNA, (iii) strong binding to the cap-binding protein eIF4E, (iv) inhibitory potential.